Novel bioactivity of furanochromene coumarin from psoralea corylifolia seeds and their synthetic analogues on skin fibroblast cells

30 Isolation, Purification and Structural Elucidation of 9H-furo [3, 2-f] chromene and coumarin derivatives from Psoralea corylifolia seeds that induce proliferation of human skin fibro

NOVEL BIOACTIVITY OF FURANOCHROMENE & COUMARIN

FROM PSORALEA CORYLIFOLIA SEEDS AND THEIR SYNTHETIC

ANALOUGES ON SKIN FIBROBLAST CELLS

SHIRIDI SAI PRASANNA M.Sc (Chemistry)

A THESIS SUBMITTED FOR THE DEGREE OF

MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2010

ACKNOWLEDGEMENTS

Firstly, I would like to express my heartfelt gratitude to National University of Singapore, for supporting me with their scholarship and facilities without which this project wouldn’t have happened

I would like to thank my Supervisor, Dr Sanjay Swarup, for giving me the opportunity to work in a very interesting area, and for his support and guidance throughout my graduate studies

A special thanks to my Co-Supervisor Dr Suresh Valiyaveettil, for his insights and constant guidance throughout the project I am very thankful to Dr Phan Toan Thang and Dr Masilamani Jeyakumar for their support and initiating this project

A million thanks to Dr Priya Ponmudi, who supported me with my experiments through the hardest times of my project I also like to thank our lab manager Liew Chye Fong for being very supportive with the administration work

My source of inspiration and strength is none other than my mother Dr Bhagavathi and my family I am greatly in debt to them for all the love and support they have given me

I would like to thank all my lab mates, especially, Weiling, Cui Ching, Amit, Sandhya, Malar, Balaji, Nizar for not only making the environment in the lab very homely but also for their help and advice

Friends! Ayshwarya, Gauri, Karthik Sheela, Satish, Sravanthy and all others for the moral support and also for the hours spent in discussing science

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

SUMMARY vii

LIST OF FIGURES ix

LIST OF TABLES xii

LIST OF ABBREVIATIONS xiii

LIST OF PUBLICATIONS xv

Chapter 1 1

Review of Literature 1

1.0 Introduction 2

1.1 Natural Product Chemistry 2

1.2 Plant-Derived Furanocoumarins and Furanochromenes: 3

1.3 General characterization of Psoralea corylifolia 8

1.4 Seed Constituent analysis 9

1.5 A link between natural and synthetic worlds 11

1.6 Mode of Action: 11

1.6.1 Synthetically - enabled Mode of Action: 13

1.6.2 A vital role for total synthesis 13

1.7 Assays for Cell Viability and Proliferation 14

1.8 Potential Application of Furanocoumarins in Wound Healing Process 15

1.8.1 Coumarin-derived natural products 16

1.9 Aims of this project 17

Chapter 2: 18

Materials & Methods 18

2.0 Introduction 19

2.1 Seed Processing 20

2.1.1 Seed Extraction 20

2.1.2 Reverse Phase High Performance Liquid Chromatography (RP-HPLC) using Analytical Column 20

2.1.3 Scale up of RP-HPLC using Semi- Preparative Column 21

2.2 Purifying and characterizing compound 8b 21

2.2.1 Extraction of solvent from compound 8b by lyophilization 21

2.2.2 Purification of compound 8b by rotavapor 22

2.2.3 Structural analysis using Mass Spectrometry 22

2.2.4 Nuclear Magnetic Resonance 22

2.3 Purifying and characterizing compound 8f mixture 23

2.3.1 Sample preparation for RP-HPLC for further analysis 23

2.3.2 Further analysis of compound 8f mixture by RP-HPLC 23

2.3.3 Further analysis of compound 8b and 8f mixture by simple, small scale thin layer chromatography (TLC) 23

2.3.4 Scale up of TLC by using Preparative TLC 24

2.3.4.1 Depositing sample 8b and 8f mixture on TLC plate 24

2.3.4.2 Identification and collection of different compound on TLC plate 24

2.4.1 Maintenance of Skin Fibroblast Cells 25

2.4.2 Seeding of fibroblast cells into 96 multiwell plate 25

2.4.3 Qualitative investigation of fibroblast cells 26

2.4.4 MTS proliferation assay 26

2.4.5 Compound 8b and compound 8f drug preparation 26

2.5 Synthesis of Furanocoumarins: (all reagents were brought from Sigma-Aldrich) 27 2.5.1 Methods for Synthesis of Furanocoumarins 27

2.5.1.1 Step 1: Ethyl coumarin-3-carboxylate derivatives 27 2.5.1.2 Step2: Ethyl 8-bromo-7-hydroxy-2-oxo-2H-chromene-3carboxylate 27

2.5.1.3 Step 3: Sonogashira reaction procedure: 27

2.6 Spectroscopic Characterization: 28

2.7 Cell Cycle Analysis 28

2.8 Confocal Imaging: 28

Chapter 3 30

Results and Discussion 30

Isolation, Purification and Structural Elucidation of 9H-furo [3, 2-f] chromene and coumarin derivatives from Psoralea corylifolia seeds that induce proliferation of human skin fibroblast cells 30

3.0 Introduction 31

3.1 Purifications of Fc-Compounds Using Reverse Phase HPLC 31

3.2 Further purification of Fc-8b and Fc-8f using preparative Thin Layer Chromatography (TLC) technique 35

3.3 Quantification of the compounds Fc-8b and Fc-8f 36

3.4 Structural elucidation of Compound Fc-8b and Compound Fc-8f 36

3.4.1 ESIMS, 1H, 13C, 2D-COSY NMR studies of FC-8b 38

3.4.2 ESIMS, 1H, 13C, 2D-COSY NMR studies of FC-8b 40

3.5 Investigation on Activity of Selected Compounds on Human Skin Cells 42

3.5.1 MTS based assay of Fc-8b on primary human skin fibroblast cells 42

3.5.2 MTS based assay of Fc-8f on primary human skin fibroblast cells 43

3.5.3: Concluding Remarks! 45

Chapter 4 46

Results & Discussion 46

Synthesis and characterisation of Furanocoumarin derivatives and their activity on skin fibroblast cells 46

4.0 Introduction 47

4.1 Rationale behind the synthesis of Furanocoumarin compounds 48

4.2 The synthetic scheme for Fc-compounds 48

4.2.1 Michael addition reaction to synthesize the coumarin moiety 49

4.2.2 Directed bromination using Br2/AcOH 50

4.2.3 Sonogashira coupling 51

4.2.4 The combined scheme to synthesize Fc-derivatives 53

4.3 Structural elucidation of Syn1 and Syn2 56

4.3.1 ESIMS, 1H, 13C, 2D-COSY NMR studies of Syn1 (5) 56

4.3.2 ESIMS, 1H, 13C, 2D-COSY NMR studies of Syn 2 (6) 58

4.4 Investigation on Activity of Syn1 (5) and Syn2 (6) compounds on skin fibroblast Cells 60

4.4.1 MTS based assay of Syn1 (5) on primary human skin fibroblast cells 60

4.4.2 MTS based assay of Syn2 (6) on primary human skin fibroblast cells 61

4.4.3 MTS-based assay of combined Fc-8b, Fc-8f, Syn1 (5), on primary human skin fibroblast cells 63

4.5 Imaging of nucleus of stained cells treated with Fc-compounds 64

4.6 Investigation on molecular effects of compound Fc-8b, Fc-8f and Syn1 on skin fibroblast 68

4.6.1 Flow Cytometry Measurement of Cellular DNA Content 68

4.6.2 Flow Cytometry analysis of the drugs 72

Chapter 5 74

Significance and Conclusions 74

5.1 Significance and final conclusion: 75

5.1.1 Stages of cell cycle 75

5.1.2: The Natural world: 78

5.1.3: Link to the Synthetic world: 78

5.1.4: Cellular effects of active compounds 79

Future Directions 80

6.1 Directions 1: Organic synthesis of Fc-derivatives 81

6.2 Direction 2: Studies on mechanism of action 82

REFERENCES 83

APPENDIX 90

SUMMARY

Structures derived from natural products have led to discovery of numerous drug leads in modern-day drug development process Here, we report a new class of compounds that we term Fc-metabolites, reported for the first time from a natural

source Complete structure of the first furanochromene (9H-furo [3,2–f] chromene;

abbreviated as Fc-8b) and furanocoumarin (abbreviated as Fc-8f) are described based

on 1H, 13C, 2D-COSY nuclear magnetic resonance spectroscopy and mass spectrometer Furanochromenes are most related to the furanocoumarins class of compounds We have developed their purification and identification procedures from

the seeds of the legume plant, Psoralea corylifolia that is found commonly in Asia, to

produce more than 99% pure compounds More significantly, we report that these metabolites are highly active in inducing proliferation of primary human skin cells at concentrations as low as 5 µM The level of proliferation induced by these compounds

Fc-is comparable to that by the universal standard 10% fetal calf serum (FCS) in the same time-period We found that therapeutically, these compounds have wide range

of applications but our initial study had few disadvantages such as, these compounds have short shelf-life period and the concentrations of the active compounds purified was very low These disadvantages led us to the second part of our study

Availability of structural information from these two bioactive Fc-metabolites shows that the furan and pyran rings are involved in isomerisation These provide a structural basis for synthesizing further synthetic bioactive furanochromenes (Fc-compounds) Based on the available structure we have developed a novel scheme to synthesize furanocoumarins and furanochromenes Using this scheme we have

synthesized two new furanocoumarin derivatives, of which one is active (Syn 1) and even better in inducing proliferation than natural forms reported here The cellular level studies show that these bioactive drugs trigger the proliferations mechanism via the G2/M phase of the cell cycle

LIST OF FIGURES

Figure 1.1: Biosynthetic coupling of Dimethylallyl pyrophosphate (DMAPP) and 7

hydroxycoumarin (umbelliferone) giving rise to furanocoumarins 4

Figure 1.2: Basic structures of Psoralen and Angelicin 5

Figure 1.3: Other furanocoumarin derivatives from natural sources .6

Figure 1.4: Biosynthesis and Structures of Furanocoumarins .7

Figure 1.5: The components of natural product research are divisible into sectors, including: isolation and structure elucidation (orange), activity screening and biological studies (red), chemical synthesis (yellow), biosynthesis (blue) and analogue preparation (green) (James J La Clair, 2009) 13

Figure 1.6: Scheme showing the reduction of MTT to formazan 15

Figure 2.1: Outline of experimental approaches to investigate the effects of selected constituents from the seeds of Psoralea corylifolia on skin cells 19

Figure 3.1 (a): RP-HPLC chromatogram of methanolic seed extracts of P corylifolia seeds X axis: elution volume (mL); y axis: UV signal intensity (arbitrary absorbance units at 254nm) The elution profile is shown in green (b): Enlarged view of the chromatogram zone containing the compounds of the bioactive fraction 8b and 8f 33

Figure 3.2: Preparative-TLC of Fc-8b and Fc-8f deposited on TLC plate 35

Figure 3.3: (a) The Ball-and-stick model of Fc-8b (b) Structure of compound 8b obtained from Mass spectrometry and NMR structural analysis (1H, 13C and 2D COSY NMR) Atom numbering differs from usual furobenzochromone numbering 37

Figure 3.4: (a) The ball-and-stick model of Fc-8f (b) Structure of compound 8f obtained from Mass spectrometry and NMR structural analysis (1H, 13C and 2D COSY NMR) Atom numbering differs from usual furobenzochromone numbering 39

Figure 3.5: MTS-based Proliferation Assay on Human Skin Fibroblast Cells (NF103, P6) upon Application of Compound Fc-8b 42

Figure 3.6: MTS-based Proliferation Assay on Human Skin Fibroblast Cells (NF103, P6) upon Application of Compound Fc-8f 43

Figure 4.1: Difference between Furanocoumarin and Furnochromene 48

Figure 4.2: Michael addition reaction showing the formation of coumarin 49

Figure 4.3: Bromination of ethoxy coumarin (product from step one) 50 Figure 4.4: Sonogashira coupling reaction 52

Figure 4.5: Three step synthetic scheme for total synthesis of Fc-derivatives The final products Syn1 (5) is shown in red and Syn2 (6) in blue 54

Figure 4.6: (a) The ball-and-stick model of Syn1 (5) (b) Structure of synthetic compound Syn1 (5) obtained from mass spectrometry and NMR spectra (1H, 13C) 55

Figure 4.7: (a) The ball-and-stick model structure of Syn 2 (6) (b) Structure of compound Synthetic Syn 2 (6) obtained from Mass spectrometry and NMR spectra (1H, 13C) 57

Figure 4.8: Effect of Syn1 (5) on proliferation of human skin fibroblast cells (NF103, P6) 60

Figure 4.9: Effect of Syn2 (6) on proliferation of human skin fibroblast cells (NF103, P6) 61

Figure 4.10: Effect of Fc-8b, Fc-8f, and Syn1 (5) on proliferation of human skin fibroblast cells (NF103, P6) 63

Figure 4.11: Confocal laser showing images of PI-stained skin fibroblast cells (a) Cells treated with DMEM lacking FCS (b) Cells treated with DMEM+10% FCS Cells treated for 48hr with (c) 5µM Fc-8b (d) 5µM Fc-8f (e) 5µM Syn1 These images are without DIC showing the contrast more clearly 65

Figure 4.12: Confocal laser showing images of PI-stained skin fibroblast cells (a) Cells treated with DMEM lacking FCS (b) Cells treated with DMEM+10% FCS Cells treated for 72hr with (c) 5µM Fc-8b (d) 5µM Fc-8f (e) 5µM Syn1 These images are with DIC to highlight the cell proliferation 66

Figure 4.13: PI stained flow cytometry assay of human skin fibroblast cells (NF103, P10) after 24hr treatment with compounds Fc-8b, Fc-8f, and Syn1 respectively 69

Figure 4.14: PI stained flow cytometry assay of human skin fibroblast cells (NF103, P10) after 48hr treatment with compounds Fc-8b, Fc-8f, and Syn1 respectively 70

Figure 4.15: PI stained flow cytometry assay of human skin fibroblast cells (NF103, P10) after 72hr treatment with compounds Fc-8b, Fc-8f, and Syn1 respectively 71

Figure 4.16: Cell Cycle regulation by Fc-8b, Fc-8f and Syn1: Propidium iodide based flow cytometric assay of cell cycle indicated divergent effects of Fc-8b, Fc-8f and Syn1 on skin fibroblast cells The phase and time-points at which the drug induces

Figure 5.1: Checkpoints in cell cycle (Roberts, J et al, 2003) 76

Figure 6.1: Derivatives that can be synthesised 81

2D-COSY 2Dimensional-Correlation spectroscopy

CDK2 Cyclin Dependent Kinase 2

CDK4 Cyclin Dependent Kinase 4

CDK6 Cyclin Dependent Kinase 6

DMEM Dulbecco’s Modified Eagle Medium

DMSO Dimethylsulfoxide

ESI Electrospray ionisation

FCS Fetal calf serum

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

NF Normal Human Fibroblast Cells

NMR Nuclar magnetic Resonance

P.I Propidium iodide

PBS Phosphate Buffered Saline

PBS Phosphate buffered saline

PUVA Psoralen ultraviolet A range therapy

RP-HPLC Reverse Phase-High Performance Liquid Chromatography

rpm Rotations per minute

SDS Sodium dodecyl sulphate

LIST OF PUBLICATIONS

PATENT

US Patent filed, US Provisional Application No.: 61/322,966 Date: 12 April 2010

Sanjay Swarup, Masilamani Jeyakumar, Shiridi Sai Prasanna, Phan Toan Thang, Suresh Valiyaveettil Furanocoumarins and Furanochromenes that Rapidly Induce Human Skin Cell Proliferation

PUBLICATIONS

Shiridi Sai Prasanna, Masilamani Jeyakumar, Thang T Phan, Suresh Valiyaveettil,

and Sanjay Swarup 9H-furo [3, 2-f] chromene and coumarin derivatives isolated

from Psoralea corylifolia seeds that induce proliferation of human skin fibroblast

cells

(Submitted to Journal of Natural Products, ACS publications, under review)

Chapter 1 Review of Literature

1.0 Introduction

1.1 Natural Product Chemistry

A natural product is a chemical compound or substance produced by a living

organism - found in nature that usually has a pharmacological or biological activity

for use in pharmaceutical drug discovery and drug design A natural product can be

considered as such even if it can be prepared by total synthesis These small molecules provide a template or skeleton for the majority of FDA-approved agents and continue to be one of the major sources of inspiration for drug discovery In particular, these compounds are important in the treatment of life-threatening conditions (Dudler, R et al 2008)

Plants have always been a rich source of lead compounds some of the classic examples are morphine, cocaine, digitalis, quinine, tubocurarine, nicotine, muscarine and didemnin Many of these lead compounds are useful drugs by themselves (e.g morphine and quinine) while others have been the basis for synthetic drugs (e.g local anaesthetics developed from cocaine) Plants provide a large bank of rich, complex and highly varied molecular structures that are unlikely to be synthesized

in laboratories Furthermore, evolution seems to have already carried out a selective process whereby plants survival increase based on the presence of anti-herbivory compounds Such products deter animals or insects from foraging on plants Despite these benefits of plant-derived compounds, the number of plants that have been extensively studied is relatively very few and the vast majority of plant species have not been thoroughly studied at all (J Merrick et al 1995)

1.2 Plant-Derived Furanocoumarins and Furanochromenes:

Furanocoumarins, or furanochromenes, are a class of organic chemical compounds produced by a variety of plants They are biosynthesized partly through the phenylpropanoid pathway and the mevalonate pathway, which is biosynthesized

by coupling of dimethylallyl pyrophosphate (DMAPP) and 7 hydroxycoumarin (umbelliferone) (Keggs, 2001) (Figure 1.1 and Figure 1.4) The first step in the bergapten biosynthetic pathway is the C-alkylation of the ortho-carbon to phenol Subsequently, a series of oxidative enzymatic reactions take place

Benzopyran is an polycyclic organic compound that results from the fusion of

a benzene ring to a heterocyclic pyran ring According to IUPAC nomenclature it is called chromene An additional furan ring attached to chromene becomes furanochromene Furanochromene also belong to the same class Furanocoumarin as both these compounds share the same chromophore

DMAPP

O O HO

+

Umbelfenone

C-alkylation at activated position

ortho to phenol

O O HO

Demethyl suberosin

O O O

OH

SAM

O O O

O O

Coumarin Structure: Coumarin is a precursor of furanocoumarins

Psoralen Structure: A linear furanocoumarin Angelicin Structure: An angular furanocoumarin.

These are precursor to many members of furanocoumarin family.

Figure 1.2: Basic structures of Psoralen and Angelicin

The compounds that form the core structure of the two most common isomers are Psoralen and Angelicin (Fig 1.3) Derivatives of these two core structures are referred to respectively as linear and angular furanocoumarins (O’Kennedy and Thornes, 1997)

Furanocoumarin derivatives

O O

O OCH3

O O

O

OCH3

O O

O

OCH3

OCH3

O O

Figure 1.4: Biosynthesis and Structures of Furanocoumarins

Many furanocoumarins are toxic and are produced by plants as a defence mechanism against various types of predators ranging from insects to mammals (Verpoorte, 1998) This class of phytochemical is responsible for the phytophotodermatitis seen in exposure to the juices of the wild parsnip

Furanocoumarins have other biological effects as well For example, in humans, bergamottin and dihydroxybergamottin are responsible for the "grapefruit juice effect", in which these furanocoumarins affect the metabolism of certain drugs (Tsai et al, 1995)

1.3 General characterization of Psoralea corylifolia

(Matsuda et al, 2007) It has been used as traditional medicine in India and China for several skin diseases such as psoriasis, leucoderma, and vitiligo (Sathe et al,

2010).Various parts of P corylifolia plant have different medicinal values For

example, the root is useful in treating the caries of the teeth and it is also used to promote bone calcification, making it useful for treating osteoporosis and bone fractures (Joshi, 2000) Leaves are used to alleviate diarrhoea and fruit extracts are used in treating piles, bronchitis, and anemias (Kong et al, 2007) The seeds are anti pyretic and alexiteric The seed extracts are known to have various anti-microbial

activities such as, inhibiting the growth of Staphylococcus citrates, Staphylococcus

antibiotics Psoralen inhibits bacteria such as Microsporium canis, Micrisporium

gypseum, Trichophyton rubrum, Trichophyton mentagrophytes, S, aureus, Candida

Psoralea corylifolia has immense biological importance to man and it has been widely exploited for its miraculous effect on several skin diseases like psoriasis, leucoderma, leprosy, and illness such as enuresis, pollakiuria, painful feeling of cold

in the waist or knees and weak kidney Studies have shown that these seeds consist of broad biological activities such as cytotoxicity, antimutagenic, bone calcination, anti-inflammatory, antibacterial, antifungal, antifeedant and antirepellant (Sah P et al, 2006) Vitiligo is an acquired cutaneous depigmentation disorder which is associated with loss of melanocytes from the basal layer of epidermis and it was reported that patients underwent six months of Psoralea treatment regained pigmentation and were cured Besides, psoralens, components in Psoralea cause residual pigmentation when applied on hypo-pigmented skin, together with increased blood flow and melanin producing activity in the affected area (Jean-paul Ortonne, 1989) In view of these numerous therapeutic effects of Psoralea seed, it is of great interest to further investigate new constituents in the seed and examine its bioactivity on skin fibroblast

1.4 Seed Constituent analysis

the main groups of constituents is coumarins which include psoralen or isopsoralen while another group is flavones which include neobavaisoflavone, corylin, bakuchiol, bavachinin, bavachin and isobavachalcone which are antioxidative

Studies have shown that the seeds of P corylifolia contain several important

chemical constituents including coumarins and flavones such as psoralen, isopsoralen, psoralidin and bavachalcone (Zhao et al, 2005) Psoralen and isopsoralen are the main

coumarins components of P corylifolia (Liu et al, 2004) Psoralia extracts and

psoralen have been used in the treatment of psoriasis The most common form of

psoriasis is chronic plaque psoriasis and is characterized by red, heavily scaled, well demarcated plaques on elbows, knees, scalp, and lower back, although any skin surface may be involved predominating in the epidermis (Cumberbatch et al, 2006)

In view of the numerous potential applications of compounds from Psoralea and presence of many modified coumarin-derivatives in Psoralia, we investigated the

effects of seed methanolic extracts and its components on human primary skin cells

Other constituents of the seeds include bovachin, bavachromene, psoralidin, isobavachalcone, bavachinin and bavachalcone It has also been reported that there are two unknown constituents of the seeds that are yet to be identified (Zhao et al., 2005b) The distribution of these constituents in the plants generally occurs at the highest level in the fruits followed by roots, stems and leaves (in order of decreasing occurrence) In addition, seasonal changes and environmental conditions may affect

the occurrence in various parts of the plant (O’Kennedy and Thornes, 1997) Psoralea

and Chinese medicine in the treatment of many diseases such asenuresis, pollakiuria, painful feeling of cold in the waist or knees and weak kidney (Zhao et al., 2005b) More importantly, it has also been used in the treatment of skin diseases such as psoriasis and vitiligo (Zhao et al., 2005a) The most common form of psoriasis is chronic plaque psoriasis and is characterized by red, heavily scaled, well demarcated plaques on elbows, knees, scalp, and lower back, although any skin surface may be involved predominating in the epidermis(Cumberbatch et al, 2006)

been used as traditional medicine in India and China for several skin diseases like psoriasis, leucoderma, vitiligo etc The objective of this work is to extract and purify compounds from the seeds, characterize it and further test their biological effects on

primary human skin fibroblast cells Here, we have identified two peaks in RP-HPLC which have immense biological effects, namely peak 8b and peak 8f Compound Fc-8b and Fc-8f were successfully characterized from peak 8b and 8f Here, compound Fc-8b has been characterized as novel chromene derivatives and Fc-8f was a novel furanocoumarin derivative and both of them, show significant effects on cell proliferation, to the levels comparable to effects of growth factors or fetal calf serum

1.5 A link between natural and synthetic worlds

Natural product research has been traditionally defined within different research sectors that include: isolation, elucidation, activity screening, chemical synthesis and biosynthesis Often these studies are conducted by independent laboratories whose research is focused on one aspect of the process, such as natural product isolation or total synthesis (J C Venter et al 2001)

This process usually begins with an extract or culture Natural product isolation is a difficult task given the complexity of marine and terrestrial extracts A number of techniques, including bioactivity-guided methods, high-throughput screening and fraction library guided approaches, now exist to index the components

of an extract While vital tools, these approaches often fail to provide feedback as to the role their structures play in delivering a unique mode of action (MOA) (E S Lander et al 2001) Related structures of natural products can help to identify active centres in the structure and guide in designing of synthetic analogues

1.6 Mode of Action:

MOA refers to an event in which a small molecule regulates a specific biological process This phenotypic effect is a complex multilevel process that has

molecular structure), molecule (as in targeting and signalling), cell, tissue, organism, and ecosystem

Within their compact uniquely organized motifs, natural products contain a

‘‘structural code’’ that allows them to target a specific biological molecule or bio- molecular interaction Regulation of these binding events is then translated into a phenotypic modification that arises either at the level of the cell (e.g., a blockage in mitosis), organism (i.e., limb development), or ecosystem (i.e., chemical defence) It

is within these fundamental connections between molecular signals and their phenotypic responses that define a molecule’s MOA The connection between these different experimental approaches (e.g., combining molecular target elucidation with cellular microscopy) adds complexity to MOA research To make matters confusing, the term mechanism of action (also MOA) is used interchangeably within the literature

Understanding the MOA of a natural product often requires a delicate balance between multiple research sectors For instance, the complement between natural isolation and chemical synthesis is key to develop analogues that allow one to further probe the activity of a compound, while activity screening and biosynthesis are key to guiding MOA studies and compound production To tackle this diversity, laboratories are faced with either expanding their research focus to integrate aspects of cell and molecular biology into their natural product and synthetic organic chemistry cores, or they develop collaborations Within the last decade, collaborative programs have provided a leading force in the elucidation of natural product activity (M E Maier, 2009) Such studies have led the way in MOA studies, as well as for methods development All the components that give a total picture of MOA are summarized in Fig 1.5 The following sections provide an overview of select studies

Figure 1.5: The components of natural product research are divisible into sectors, including: isolation and structure elucidation (orange), activity screening and biological studies (red), chemical synthesis (yellow), biosynthesis (blue) and analogue preparation (green) (Clair, 2009)

1.6.1 Synthetically - enabled Mode of Action:

In addition to advances in isolation, elucidation and biological evaluation, support for synthetic organic chemistry is vital to the further understanding of natural product biology The following section provides a brief overview of the interplay between chemical synthesis (total synthesis) and the understanding of natural product activity

1.6.2 A vital role for total synthesis

While the synthetic community often focuses on the development of chemical methods and synthetic approaches, total synthesis also has several fundamental roles

in MOA science First, it serves as a vital tool to confirm (or revise) the structural assignments (Butler, M.S 2008) Second, it provides access to materials that may not

be readily available Finally and perhaps most importantly, it provides access to probe and probe classes that cannot be accessed naturally The importance of MOA work

has become a common theme in natural product total syntheses, with a large majority

of the total syntheses in the period of this review stating the importance of providing access to subsequent MOA and activity studies; salient examples include amphodinolides, (+)-acutiphycin, the bryostatins, chlorosulfolipid cytotoxins, diazonamides etc (Newman, D, J and G M Cragg, J 2007)

1.7 Assays for Cell Viability and Proliferation

Cell proliferation assays are particularly valuable in medical research and drug development Examples include chemically induced mutagenicity and carcinogenicity assays, the screening of novel growth and differentiation factors and the detection of oestrogenic compounds (Kintzios, 2005) In this project, tetrazolium-based colorimetric assay (MTS test) has been used to determine the cell proliferation under

influence of specific compound found in Psoralea corylifolia seeds

The in vitro MTS test was first described by Promega which is a modification

of the MTT assay, to detect mammalian cell survival and proliferation It is a rapid colorimetric method based on the cleavage of yellow tetrazolium salt (3-{4, 5-dimethylthiazol-2-yl}-2, 5-diphenyl tetrazolium bromide) to purple formazan precipitate by mitochondrial dehydrogenases enzyme of metabolically active cells Given that the total amount of formazan generated in the plate is directly proportional

to the number of viable cells, this method has been adopted largely for its rapidity and accuracy in measuring cell survival and proliferation (Fig 1.6) (G Ciapetti et al, 1993)

Figure 1.6: Scheme showing the reduction of MTT to formazan

However, when applying MTT test, several cautionary steps need to be observed as catechols and flavonoids, quercetin

serum supplemented medium that could resulted in false readings on the spectrophotometer Antioxidant effects of plant extracts such as

fibroblast cells were also observed (Phan et al 2000) These issues

using MTS reagent It must be noted that MTT and MTS have the same chemical structure MTS assay reagent contains

all other constituents found in MTT assay reagent

1.8 Potential Application of Fu

Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers The human adult wound healing process can be divided into 3 distinct phases: the inflammatory phase, the proliferative

remodeling phase Within these 3 broad phases is a complex and coordinated series of events that includes chemotaxis, phagocytosis, neocollagenesis, collagen degradation, and collagen remodeling

Scheme showing the reduction of MTT to formazan

However, when applying MTT test, several cautionary steps need to be observed as catechols and flavonoids, quercetin were found to reduce the MTT in serum supplemented medium that could resulted in false readings on the

spectrophotometer Antioxidant effects of plant extracts such as Buddleja globosa

fibroblast cells were also observed (Phan et al 2000) These issues are removed on

It must be noted that MTT and MTS have the same chemical MTS assay reagent contains phenazine methosulfate (PMS) in addition to all other constituents found in MTT assay reagent

Potential Application of Furanocoumarins in Wound Healing Process

Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers The human adult wound healing process can be divided into 3 distinct phases: the inflammatory phase, the proliferative phase, and the remodeling phase Within these 3 broad phases is a complex and coordinated series of events that includes chemotaxis, phagocytosis, neocollagenesis, collagen degradation, and collagen remodeling

However, when applying MTT test, several cautionary steps need to be

were found to reduce the MTT in serum supplemented medium that could resulted in false readings on the

are removed on

It must be noted that MTT and MTS have the same chemical

(PMS) in addition to

ranocoumarins in Wound Healing Process

Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers The human adult wound healing process can be divided

phase, and the remodeling phase Within these 3 broad phases is a complex and coordinated series of events that includes chemotaxis, phagocytosis, neocollagenesis, collagen degradation,

In diabetic wounds or diabetic ulcers, however, healing impairment occurs One important characteristic of diabetic wounds in relation to this project is that some

of the resident cells in diabetic foot ulcers become phenotypically altered Fibroblasts isolated from diabetic foot ulcers are probably senescent and show a decreased proliferative response to growth factors (Loots et al, 2002) Evidence suggests that phenotypic changes in wound cells are not due only to replicative senescence, but are perhaps caused by more complex interactions between the resident cells and the chronic wound Although there is no direct evidence that the proliferative activity of keratinocytes is affected in diabetes, migration of these cells is likely to be impaired (Falanga, 2005)

Therefore, the availability of natural product derived compounds that can increase proliferation in human skin cells such as fibroblast and keratinocytes may be therapeutically useful in the process of wound healing, not just in healing diabetic wounds, but also other types of wounds such as burns

1.8.1 Coumarin-derived natural products

Coumarins are often found as secondary metabolites that play role in plant defense due to various stress events in plants (Zobel, 1997) They belong to benzopyrones, a group of compounds where a benzene ring is joined to a pyrone A pyrone is a six member heterocyclic ring, containing one oxygen atom and a five sp2 hybridized carbons This compound has numerous bioactivities and serves as skeleton for a number of commercially available drugs used for psoriasis, anticoagulants, high protein oedema and many other conditions (O’Kennedy and Thornes, 1997) In spite

of all these positive effects, there are no reports of mitogenic action which is crucial in cell division and proliferation

Furanocoumarins such as xanthotoxin and bergapten, whose biogenetic precursor is umbelliferone, belong to a subclass of the coumarin family It consists of

a five member furan ring attached to the coumarin nucleus which can be either psoralen or angelicin Different ways and angles of furan binding to the core structure will then give rise to different isomers

Furanocoumarins are products of the shikimate pathway which is the biosynthetic route for aromatic amino acids biosynthesis and this subclass of compounds are derived from precursors such as coumaric acid and umbelliferone

‘Linear’ furanocoumarins were form when the furan rings in line with the pyrone nucleus; while ‘angular’ furanocoumarin are those where the furan ring is oriented at an angle to the nucleus Furanocoumarins are of pharmaceutical interest because of their numerous biological activities and this compound plays an important role in natural compound-derived drugs in therapies as skin repigmentation stimulants

benz-2-1.9 Aims of this project

This project aims to extract, separate, identify and purify novel compounds

found in seeds of Psoralea corylifolia These compounds were characterized and

tested for their biological activities on human primary skin cells A synthetic pathway was designed and used to synthesize the bio-active forms of naturally isolated compounds A comparative bioactive study was done to see if the synthetic compounds emulate similar activity as naturally isolated compounds Further, cell cycle analysis using flow cytometry and confocal imaging were done to identify the cell cycle stage that was affected and visualise the proliferated cells

Chapter 2:

Materials & Methods

2.0 Introduction

The Materials and Methods section encompasses detailed descriptions of all

the methods employed and the protocols followed while performing the experiments

It also provides the source of all reagents and biological materials used in the

research

Figure 2.1 summarizes the experimental approaches applied in this project in

order to investigate the effects of the selected constituents of the seeds of Psoralea

Figure 2.1: Outline of experimental approaches to investigate the effects of selected constituents from the seeds of Psoralea corylifolia on skin cells

2.1 Seed Processing

2.1.1 Seed Extraction

India 100mg of seeds were frozen in liquid nitrogen and ground into fine powder, and then homogenized in 1ml of 80% methanol (HPLC graded) to extract the hydrophobic metabolites This homogenized mixture was then centrifuged at 14,000rpm for 30minutes at 4°C Supernatant was collected and filtered using 0.2µ filter to obtain homogenous crude sample The samples were stored at 4°C in darkness

2.1.2 Reverse Phase High Performance Liquid Chromatography (RP-HPLC) using Analytical Column

The HPLC system used was ÄKTA purifier TM 10 from Amersham Biosciences In this chromatographic analysis, Jupiter TM 5µm C18 300 Å analytical column with the dimension of 250 x 4.6mm from Phenomenex, Inc., USA, was used 100µL of methanolic crude sample was injected for the analytical run A linear gradient elution method using two buffers: Buffer A (water-acetic acid at pH3; 5ml/250ml of water) as aqueous solvent and Buffer B (100% Acetonitrile) as organic solvent, was carried out All buffers that were used in HPLC must be HPLC graded and filtered with membrane filter with a pore width of 0.2 µm An elution profile with A: B ratio – 0 min 100:0; 60 min 40:60; 66min 0:100 were performed Following that, isocratic elution with 100% Acetonitrile was carried out for 5 min The flow rate was 0.5ml/min and the compounds were detected at an excitation wavelength of 254nm

2.1.3 Scale up of RP-HPLC using Semi- Preparative Column

Fivefold scale up was carried out using Jupiter TM 5µm C18 300 Å with dimension 250 x 10.00 mm semi-prep column The HPLC system used was ÄKTA purifier TM 10 from Amersham Biosciences Here, volume injected for the whole run was 900µL but it breaks down to a total of 9 injections because the loading loop capacity was 100 µL A linear gradient elution method using Buffer A (water-acetic acid at pH3) and Buffer B (100% Acetonitrile) was carried out in the following A: B ratio 0min, 100:0 ; 50min, 60:40 ; 95min, 52:48 ; 105min, 36:64 ; 115min, 0:100 ; 118min, 0:100 ; 118.5min, 100.0; 120min, 100:0 The flow rate was increased to 2.363ml/min and the detection wavelength of 254nm was set During elution, fractions of 1ml per tube were collected between 52min and 55min of elution (collection of compound A) and then 1.5ml per fraction (tube) between 72 min and 96min (collection of compounds from 8a to 8f) Fractions of each peak that were collected were labelled and store in 4’C fridge under dark conditions

2.2 Purifying and characterizing compound 8b

2.2.1 Extraction of solvent from compound 8b by lyophilization

The fraction 8b collected from HPLC fraction from ~78min to 81min time point was lyophilized to get rid of the solvent which were acetic acid and acetonitrile The samples were collected in falcon tube, filling it up to one third of the tube capacity Then, the tubes were freeze at -80°C fridge in an inclined position at about 140° until solidification After freezing the samples, they were sent for lyophilization

at Protein and Proteomics Center (NUS) The freeze-dry process took at least 15hours

to obtain the pure solid 8b compound

2.2.2 Purification of compound 8b by rotavapor

Because each tube that sent for lyophilization will only consist of ~15mL of fraction 8b, amount of solid form of 8b that were collected were minute and ‘sticked’

on tube surface All these pure solid form of 8b have to be concentrated in one sample tube in order to perform further characterization test such as mass spectrometry and NMR Samples were ‘washed’ in filtered methanol (about 5mL per tube) to dissolve all solid 8b sample These entire dissolved samples were then collected in a 25mL round bottom flask and underwent rotavapor, setting temperature at 40-45°C to extract excess methanol The process will be stopped when dry samples which are brownish yellow were seen

2.2.3 Structural analysis using Mass Spectrometry

Solid sample were then dissolved in 1ml of MeOD4 and sent for mass analysis with electronspray ionization (ESI) source Mass spectra were acquired by a Micromass VG7035 double focusing mass spectrometer of high resolution The ionization mode selected was positive The masses of positive ion species within the range of 0- 500 amu were detected in ESI-MS experiments

2.2.4 Nuclear Magnetic Resonance

10mg of pure solid compound 8b was used for NMR structural analysis to examine its chemical environment Four different types of analysis which are 1H NMR, 13C NMR, COSY, and HMQC were carried out NMR spectra were recorded

on a Bruker AMX 300 spectrometer The solvent used were DMSO-d6, MeOD4

2.3 Purifying and characterizing compound 8f mixture

2.3.1 Sample preparation for RP-HPLC for further analysis

Sample obtained from HPLC fractions 8f were lyophilized as described in Section 2.2.1 Solid form (yellow powder) compound 8f mixtures were dissolved in filtered methanol as described in Chapter 3

2.3.2 Further analysis of compound 8f mixture by RP-HPLC

In this chromatographic analysis of compound 8f mixture, Synergi Polar TM

RP 4µm 80 Å, (250 x 4.6mm) analytical column obtained from Phenomenex Company were used and was performed on ÄKTA purifier TM 10 from Amersham Biosciences With this column, many optimization trials were carried out to get the perfect elution profile with highest resolution The injection volume for each analytical run is 100µL For aqueous mobile phase, water-acetic acid pH3 were used; while different mixture ratio of methanol and acetonitrile were used for organic mobile phase In this optimization process, the elusions were started with 100% methanol and then increasing addition of acetonitrile was tried Besides optimizing the mobile phase as one of the parameter, different flow rate were also been tested so that the time required for separation of 8f mixture can be as short as possible

2.3.3 Further analysis of compound 8b and 8f mixture by simple, small scale thin layer chromatography (TLC)

Sample Fc-8b and Fc-8f were deposited as a spot on the stationary phase which is 5 X 2cm silica gel plate Combination of 70% hexane and 30% of ethyl acetate were used as mobile phase to provide adequate selectivity for the 8f mixture to

be separated When the solvent reached the front, the plate was visualized under a UV

lamp For better visible result, the plate was placed in iodine chamber for 8-10 minutes

2.3.4 Scale up of TLC by using Preparative TLC

2.3.4.1 Depositing sample 8b and 8f mixture on TLC plate

Silica gel 60 F

254 preparative TLC plates (20×20 cm with 4×20 cm concentration zone, 0.5 mm layer thickness and fluorescence with excitation 254 nm) (Merck) was used as the stationary phase in this preparative TLC A band of 8f sample pre-dissolved in methanol was deposited on the plate with a glass pipette Then, hexane: ethyl acetate in the ratio 70:30 (v/v) was used as the mobile phase This preparative TLC were carried out in flat bottomed 20x 20cm TLC chamber and 100mL of mobile phase solvent were poured in Then, the 20x 20 TLC plate with sample were placed carefully inside the chamber and the separation process started Throughout the whole run, careful inspection was done with the aid of UV lamp to monitor the separation and band length of each compound found in 8f mixture

2.3.4.2 Identification and collection of different compound on TLC plate

Three bands were detected on the preparative TLC plate Then blue fluoresce band which has longer band length were identified under aid of UV light and scraped out using a TLC plate scrapper The flakes were then collected in a 250mL con flask and dissolved with dichloromethane to extract the interest compound Sonification method was used as well to ‘knock-out’ compound that bound to the silica gel Then, the silica-8f compound mixture was filtered and sent for rotavapor to extract dichloromethane